1. Field of the Invention
This invention relates to the luminesence detection and measurement art, and, more particularly, to a high speed luminescence detection arrangement for sequentially measuring the luminescence from a plurality of luminescence emitting samples.
2. Description of the Prior Art
As a method of precise analysis in both clinical and research laboratories, Luminescence Immunoassay (LIA) offers extraordinary potential. LIA combines the specificity of immunoassay and the sensitivity of luminescent light detection. Moreover, applications are almost limitless in that all that is needed is a procedure to link a luminescence reactant, such as isoluminol or peroxidase, to the analyte or its antibody availability of antibody to the analyte; and an instrument to measure the luminescent light emitted.
Both bioluminescent (BL) and chemiluminescent (CL) reactions can be utilized in these assays. Bioluminescent reactions are enzymatically mediated by bacterial or firefly luciferase, and involve oxidation of a substrate (usually luciferin) to form products which include light. Luciferase-bound analytes or ATP can be quantitated by correlating the amount of light produced to concentrations of standards. Detection limits of 10.sup.-16 mol ATP, 10.sup.-18 mol TNT, and 10.sup.-17 mol DNP are reported.
Chemiluminescent reactions involve a similar light production, commonly by oxidation of a phthalhydrazide derivative, such as luminol, in the presence of hydrogen peroxide and peroxidase. With naphthalhydrazide and phthalhydrazide derivatives as chemiluminescent labels, detection limits in the range of 10.sup.-16 mol are reported. Analytes include digoxin, thyroxine, IgG, cortisol, insulin, progesterone, and alpha-fetoprotein.
While there have been many procedures offering the specificity inherent to antibody binding, until the development of LIA, radioactive labelling (RIA) was the method of choice due to its sensitivity. LIA has all of the advantages of RIA, including comparable or better sensitivity. In addition, it features low reagent volumes (hence low cost), and nontoxic, stable reagents. Luminescence reagents offer a shelf life of about 2 years as compared with .sup.125 I, a common RIA label, which has an effective shelf life of about thirty (30) days.
The clinical chemist is involved in measuring a variety of substances by many different analytical techniques. Although different, these techniques share the common principle of an interface between chemistry and physics. The most commonly used such interface in clinical chemistry is absorptiometry, both at visible and ultraviolet wavelengths, but emission flame photometry and radioactivity are also commonly used.
Analyses based on the measurement of emitted light, such as luminescent light, offer several advantages over conventional techniques: high sensitivity, wide linear range. low cost per test, and relatively simple and inexpensive equipment.
Luminescence detection has application in several areas of clinical analysis. It has a role as a replacement for conventional colormetric or spectrophotometric indicator reactions in assays for substrates of oxidases and dehydrogenases. In this type of assay the sensitivity of the luminescence indicator reaction may be used either to quantitate substrates not easily measured by conventional techniques (e.g., prostaglandins and vitamins) or to reduce the quantities of specimen and reagent required in the initial enzymatic step, thus reducing the cost of the assay. Another application of luminescence is the utilization of luminescent molecules as replacements for radioactive labels in immunoassay, as noted above.
An important feature of the luminescence as an analytical technique is that its usefulness is not confined to clinical chemistry. Further, luminescence detection has applications in roles in other pathology disciplines, e.g., hematology, immunology, bacteriology, and pharmacology.
Chemiluminescence may be simply defined as the chemical production of light. In the literature it is often confused with fluorescence. The difference between these two processes is the source of energy that is producing molecules in an excited state. In chemiluminescence, this is the energy of a chemical reaction, and the decay from the excited state back to the ground state is accompanied by emission of light (luminescence). In contrast, incident radiation is the source of the energy that, in fluorescence, promotes molecules to an excited state. Analytically, the types of luminescence that have engendered the most interest are chemiluminescence and bioluminescence. The latter is the name given to a special form of chemiluminescence found in biological systems, in which a catalytic protein increases the efficiency of the luminescent reaction. Indeed, in certain cases the reaction is impossible without a protein component.
Of the several advantages of luminescent methods over their conventional counterparts, their extreme sensitivity is the most important. For example, as compared with spectrophotometry, the BL assay of NADH is estimated to be some 25,000-fold more sensitive, and BL assays for glucose and alcohol are, respectively, 55- and 10-fold more sensitive than conventional assays. The minimal detectable concentration for an assay ultimately depends on how sensitively light can be detected, and on the quantum efficiency of the reaction. Generally BL reactions are much more efficient than CL reactions; typical quantum efficiencies are in the ranges 0.1-0-8 and 0.01-0.05, respectively.
Since CL is an emission process (as opposed to absorption), response is usually linearly proportional to concentration from the minimal detectable concentration up to the point where it is no longer possible to maintain an excess of other reactants relative to the analyte. In the case of ATP assay by the firefly reaction, response is linear over six orders of magnitude.
The speed of analysis largely depends on the type of luminescent reaction. In some instances a rapid flash is obtained (1 s), the peak height of which may be related to analyte concentration; in other cases a more protracted glow occurs when a time course lasting several minutes. In the latter case, the integral or partial integral of the light-time curve has been used as a measure of analyte concentration because it is much less sensitive to mixing efficiency, but this drastically reduces the through-put and speed of analysis.
The major cost benefit of luminescent assays arises from their extreme sensitivity, which allows assays to be performed with much less reagents, hence reducing the cost per test. For example, luminescence offers a means of reducing the cost of cholesterol assays involving cholesterol oxidase (EC 1.1.3.6), because the sensitivity of CL detection of peroxide allows the initial peroxide-producing reaction involving cholesterol oxidase to be scaled down.
Specificity is conferred on BL and CL by using the luminescence as an indicator reaction coupled to intermediates (such as ATP, MADH, and H.sub.2 O.sub.2) produced enzymatically. Generally, BL reactions are specific because they are enzymic processes, but CL reactions are nonspecific. Luminol, for example, will undergo a CL reaction with various oxidants (oxygen, peroxide, superoxide, iodine) and its reactions are subject to interferences by reducing agents such as uric acid.
In practical applications of the principles of bioluminescence and chemiluminescence as above described, it is necessary to provide a detection and measuring arrangement for detecting and measuring the luminescence emitted from the sample under test. Further, the arrangement should include a display for displaying the signal indicative of the intensity of the luminescence emitted. One prior art device, utilized for such luminescence detection measuring applications, required that each separate sample under test be separately placed into the device. Reagents necessary to achieve luminescence with the sample under test could be added prior to insertion of the sample into the device or after insertion. The luminescence from the one sample was then detected and measured, and a display indicative of the amount of luminescence was provided. The sample was then removed and another sample could then be installed. Such sampling on a one-at-a-time basis was time consuming, and did not lend itself to applications requiring high speed, rapid, determination of the luminescence emitted from a plurality of samples.
Accordingly, there has long been a need for a luminescence detection measuring arrangement in which a plurality of samples under test may be rapidly subject to measurement of the luminescence emitted therefrom and a display, indicative of the intensity of the luminescence from each separate sample under test be provided.